Incomplete healing of bone defects ih the craniofacia.l skeleton is common. Osteogenic proteins, including bone morphogenetic protein 2 and 4 (BMP2, BMP4), promote healing in bone defects, but the proteins'short half-lives and rapid clearance by the bloodstream limit their utility. The main goal of our initial R01 and the first competitive renewal project was the development of tissue engineering approach'es, based on muscle-derived stem cells (MDSCs), to efficiently deliver osteogenic proteins and improve craniofacial bone healing. In brief, during this funding period, we demonstrated that MDSCs genetically engineered to express BMP2 and BMp4 differentiate toward an osteogenic lineage and can improve bone healing in calvarial and long bone defects. We also found that concomitant expression Of vasCular endothelial growth factor (VEGF) improves the bone healing observed after implantation Of BMP2 and 13MP4 expressing MDSCs. Additionally, we have demonstrated that donor sex influences the in vitro osteogenic potential and in vivo bone regeneration potential of murine MDSCs and also identified wa.ys, such as genetic engineering and manipulation of the BMP signaling pathways, to improve the osteogenic potential of MDSCs. Finally, we have isolated the human equivalents of the murine MDSCs and determine their osteogenic potential in vitro. We would like to thank NIDCR for their support during the prior funding period. We met and exceeded all the key objectives in the original R01 application as well as the first competitive renewal, and our results formed the basis for 33 + papers and 117+ abstracts. This DE013420 second cornpetitive renewal application outlines experiments designed to extend these initial findings and lead to. possible future clinical applications of MDSCs to improve bone healing. We will focus this second competitive renewal on human equivalents to murine MDSCs and optimization of their use for bone regeneration. Since after implantation of MDSCs into injured musculoskeletal tissues, including bone, the repair process is often mediated by chemoattraction of host cells, we plan to determine the influence of host cells(especially blood vessel wall progenitors) chemoattraCted by donor human cells during the bone healing process (Aim 1). We plan to examine the effect of age and sexotdonor patient on the number and osteogenic potential of hMDCs derived from that patient. We then will investigate ways to optimize bone formation and healing by using hMDC-based tissue engineering, including the modulation of BMP signaling through inhibition of ERK1/2, pj8 MAPK and PI3K pathwa.ys and mechanical stim'ulation of hMDCs prior to implantation (Aim 2). The proposed experiments will provide important information regarding the basic biology of hMDCs and their use for bone healing and further the development of clinical treatments for osseous deficiencies.